The precise wiring of the vertebrate nervous system depends on complex molecular cues to direct migrating axons toward synaptic targets. Primary motor neuron (PMN) development in the zebrafish provides a relatively simple, elegant model for the study of axon guidance. Uncovering the mechanisms by which individual PMNs project axons to dorsal, ventral, and lateral muscle tissue can provide valuable insight for understanding how complex neuronal networks develop in higher vertebrates. Recent studies in D. melanogaster and C. elegans have shown that glypicans are critically important for axon patterning and synapse development. Glypicans are a class of heparan sulfate proteoglycan tethered to the cell membrane through a GPI-anchor. A large variety of signaling molecules, such as Wnts, Hhs and BMPs, depend on glypicans to facilitate their distribution and signaling activity. Evidence from fruit fly, mouse and human has shown that Notum, a novel secreted enzyme, inhibits Wnt by shedding glypicans from the cell surface. However, it is unclear whether Notum similarly plays a role in axon patterning through the cleavage of glypicans. The goal of this study is to determine the role of Notum in axon guidance during zebrafish development. Our preliminary gain and loss of function assays have led us to hypothesize that Notum regulates glypicans to promote axon growth. Furthermore, we predict that Notum 2 uses a conserved mechanism of glypican shedding to release Wnt inhibition and promote axon growth. We will test this hypothesis by using gain and loss of function assays to determine how individual neurons respond to changing Notum levels in vivo. Second, we will perform a highly focused candidate screen to determine whether Notum interacts with glypicans to promote axon growth. Lastly, we will determine whether Notum interacts with a select group of Wnts with established roles in axon guidance. Though not all aspects of neuron development are analogous between fish and mammals, the molecular mechanisms of axon guidance do appear to be highly conserved. By elucidating the role of Notum in fish, we hope to expand our understanding of the molecular players involved in axon guidance in vertebrates. ) PUBLIC HEALTH RELEVANCE: During development, neurons send axonal projections to distant targets. The precise wiring of the vertebrate nervous system depends on complex guidance cues. The goal of this study is to determine the role of a novel secreted enzyme, Notum, in axon guidance during zebrafish development. By elucidating the role of Notum in fish, we hope to expand our understanding of the molecular players involved in axon guidance in vertebrates. )